Recombinant influenza virus-like particles (VLPs) produced in transgenic plants expressing hemagglutinin

Abstract

A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

What is claimed is: 
     
       1. A method of producing an influenza virus like particle (VLP) in a plant comprising:
 a) introducing a nucleic acid comprising a nucleotide sequence encoding an influenza hemagglutinin (HA) into a plant, or portion of a plant, the HA being operatively linked to a regulatory element that is operative in a plant and wherein the regulatory element comprises a Cowpea Mosaic Virus (CPMV) regulatory region, and 
 b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLP. 
 
     
     
       2. The method of  claim 1 , wherein following the step of introducing, the nucleic acid is either transiently expressed in the plant; or stably expressed in the plant. 
     
     
       3. The method of  claim 1 , wherein in the step of introducing, a second nucleic acid comprising a nucleotide sequence encoding one or more than one chaperone proteins is introduced into the plant. 
     
     
       4. The method  claim 1 , wherein after the step of incubating, the plant or portion of the plant is harvested, and the VLP purified, wherein the VLP range in size from 80-300 nm. 
     
     
       5. A plant comprising a nucleic acid comprising a nucleotide sequence encoding an influenza hemagglutinin (HA), the HA being operatively linked to a regulatory element that is operative in a plant, wherein the regulatory element comprises a Cowpea Mosaic Virus (CPMV) regulatory region; and the plant further comprising influenza virus like particle (VLP). 
     
     
       6. The plant of  claim 5 , further comprising a nucleic acid comprising a nucleotide sequence encoding one or more than one chaperone proteins operatively linked to a regulatory region active in a plant. 
     
     
       7. A method of producing an influenza virus like particle (VLP) in a plant comprising:
 a) providing a plant or a portion of a plant comprising a nucleic acid comprising a nucleotide sequence encoding an influenza hemagglutinin (HA), the HA being operatively linked to a regulatory element that is operative in a plant and wherein the regulatory element comprises a Cowpea Mosaic Virus (CPMV) regulatory region, and 
 b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLP. 
 
     
     
       8. The method  claim 7  wherein after the step of incubating, the plant or portion of the plant is harvested and the VLP is purified. 
     
     
       9. The method of  claim 1 , wherein in the step of introducing, the HA comprises a native or a non-native signal peptide. 
     
     
       10. The method of  claim 9 , wherein the non-native signal peptide is a protein disulfide isomerase signal peptide. 
     
     
       11. The method of  claim 1 , wherein in the step of introducing, the HA is a type A influenza, a type B influenza, or is a subtype of type A influenza of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. 
     
     
       12. The method of  claim 1 , wherein the influenza hemagglutinin (HA) has 70%-100% sequence similarity with the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76. 
     
     
       13. A VLP produced by the method of  claim 1 . 
     
     
       14. The VLP of  claim 13 , wherein the VLP comprises one or more than one lipid derived from a plant. 
     
     
       15. The VLP of  claim 13 , wherein the HA comprise plant-specific N-glycans or modified N-glycans. 
     
     
       16. The VLP of  claim 13 , wherein, the HA is a type A influenza, a type B influenza, or is a subtype of type A influenza of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. 
     
     
       17. A composition comprising an effective dose of the VLP of  claim 13 , and a pharmaceutically acceptable carrier. 
     
     
       18. A plant extract comprising a VLP produced by the method of  claim 1 . 
     
     
       19. The plant extract of  claim 18 , wherein the VLP is suitable for oral administration. 
     
     
       20. A food supplement comprising the plant extract of  claim 18 . 
     
     
       21. A composition comprising a plant extract comprising a VLP produced by the method of  claim 1 , and a pharmaceutical acceptable carrier. 
     
     
       22. A plant comprising a VLP produced by the method of  claim 1 . 
     
     
       23. A VLP produced by the method of  claim 7 . 
     
     
       24. The VLP of  claim 23 , wherein the VLP comprises one or more than one lipid derived from a plant. 
     
     
       25. The VLP of  claim 23 , wherein the HA comprise plant-specific N-glycans or modified N-glycans. 
     
     
       26. The VLP of  claim 23 , wherein, the HA is a type A influenza, a type B influenza, or is a subtype of type A influenza of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. 
     
     
       27. A composition comprising an effective dose of the VLP of  claim 26 , and a pharmaceutically acceptable carrier. 
     
     
       28. A plant extract comprising a VLP produced by the method of  claim 7 . 
     
     
       29. The plant extract of  claim 28 , wherein the VLP is suitable for oral administration. 
     
     
       30. A food supplement comprising the plant extract of  claim 28 . 
     
     
       31. A composition comprising a plant extract comprising a VLP produced by the method of  claim 7 , and a pharmaceutical acceptable carrier. 
     
     
       32. A plant comprising a VLP produced by the method of  claim 7 . 
     
     
       33. The method of  claim 3 , wherein the one or more than one chaperone proteins is Hsp40 and/or Hsp70. 
     
     
       34. The plant of  claim 6 , wherein the one or more than one chaperone proteins is Hsp40 and/or Hsp70. 
     
     
       35. The method of  claim 11 , wherein the subtype of type A influenza is H1, H2, H3, H5, H6, H7, or H9. 
     
     
       36. A method for inducing immunity to an influenza virus in a subject comprising administering a composition comprising an effective dose of the VLP of  claim 13  and a pharmaceutically acceptable carrier to said subject. 
     
     
       37. A method for inducing immunity to an influenza virus in a subject comprising administering a composition comprising an effective dose of the VLP of  claim 23  and a pharmaceutically acceptable carrier to said subject. 
     
     
       38. The method of  claim 1 , wherein the influenza hemagglutinin (HA) has 90%400% sequence similarity with the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 or SEQ ID NO: 59.